The present invention relates to a method for the propagation of lytic organisms, and its use for the production of lytic organisms and proteins.
A number of different organisms can be grown in tissue culture for use in research studies or vaccine production. However, the titres obtained in tissue culture, although suitable for research purposes, are often too low to be commercially useful in the vaccine industry, so bulk preparations of vaccines (e.g. flu vaccine) are often cultured in chicken eggs. This system provides very high titres of virus but requires a large amount of processing and quality control of product before release. The process is very labour intensive and has problems with continuity of supply and reproducibility of product from batch to batch. Hence there is a need for large scale, reproducible, high titre production of organisms.
Another area that feels a critical need for large scale high titre reliable virus production is gene therapy. Recombinant adeno-associated virus vectors have recently been shown to be efficient, non-immunogenic and persistent vectors for gene therapy. However, current technologies are unable to produce the amounts of recombinant viruses needed for this field to move forward (Linden. R. M and Woo, S. L. C. Nature Medicine 5(1) 1999 pp 21-22.)
The culture of lytic organisms is particularly difficult as these quickly destroy the cell population they are grown in. The insect virus baculovirus is a lytic organism which can be used for the production of proteins. These viruses can be engineered for recombinant protein expression (Gruenwald, S. and Heitz, J. Baculovirus expression vector system: Procedures and methods manual. Pharmingen). They are generally grown in spinner culture, in which maximum infection and hence maximum protein production is reached after two days. A longer term culture of baculovirus would produce greater amounts of protein, and be extremely useful but, until now, has not been possible. Also, a continuous culture would provide a sustained supply of product, which until now has only been possible with batch processes requiring repeated cultures.
The present invention provides a method for the propagation of lytic organisms which comprises the infection of the cells of a stable cell line within a hollow fibre bioreactor with a lytic organism, wherein after said infection, said organism multiplies within the cell line and can be harvested, characterised in that the cell line can survive for at least ten days after infection.
The invention further provides a method as described above, wherein the lytic organism contains nucleic acid encoding a protein of interest, and after said infection this protein is expressed by the cells and can be harvested.
Further provided is any of the methods as described herein which further comprises the step of harvesting of either the lytic organism or the expressed protein.
In another aspect of the invention is provided a method as described wherein after harvest, the cell line is allowed to re-populate the bioreactor, and at least one subsequent harvest may be taken, with the cell line being able to re-populate the bioreactor after each harvest. In this aspect the method is applicable both when the desired product is the lytic organism itself, or an expressed protein of interest.
Further provided is a method for studying the effects of molecules on a lytic organism which comprises the infection of the cells of a stable cell line within a hollow fibre bioreactor, wherein after said infection, varying amounts of said molecules may be added, and their effects on the lytic organism measured, characterised in that said cell line can survive for at least 10 days after infection.